Analytica Chimica Acta 436 (2001) 253–263

Determination of selenoethionine by flow injection-hydridegeneration-atomic absorption spectrometry/high-performance

liquid chromatography-hydride generation-high power nitrogenmicrowave-induced plasma mass spectrometry

Amit Chatterjee∗, Y. Shibata, A. Tanaka, M. MoritaEnvironmental Chemistry Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 3050053, Japan

Received 22 August 2000; received in revised form 9 February 2001; accepted 5 March 2001

Abstract

A simple direct flow injection hydride generation atomic absorption spectrometric method was developed using sodiumtetrahydroborate and hydrochloric acid for the determination of selenoethonine (Seet) that excluded any chemical pretreatmentprior to hydride generation. The detection limit (3σ of blank) of the method was 0.25 ng ml−1 selenium (standard solutionsin Milli-Q water). The reproducibility (R.S.D. of three analyses performed on three different days) and the repeatability(R.S.D. of seven successive analyses) of the method were 9.2% for 5.05 ng ml−1 and 2.8–9.5% for 20.0–1.00 ng ml−1 ofstandard selenium (Milli-Q water), respectively. The calibration graph was linear up to 20.0 ng ml−1. This HG methodis very promising and was successfully applied for the on-line estimation of Seet (spiked) in human urine with usinghigh-performance liquid chromatography-hydride generation-high power nitrogen microwave-induced plasma mass spec-trometry (HPLC-HG-N2-MIP-MS). The separation was performed on a GS-220 gel-permeating column with a 25 mMtetramethylammonium hydroxide + 25 mM malonic acid buffer (pH 7.5), which showed 26–30% signal suppression dueto less volatile hydride formation from Seet among the mobile phases examined. The recovery of Seet that was preparedin the above mobile phase is about 74% as that of the Seet prepared in Milli-Q water. In the PRP-X100 anion-exchangecolumn with the 15 mM phosphate (pH 7.0) mobile phase, Seet eluted out after 600 s keeping a broad peak with enlarg-ing the baseline signal. The concentration of selenite found in the human urine was 3.6 ± 0.2 �g l−1 and that agreed wellwith the high-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) value(3.8 ± 0.2 �g l−1). © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Selenium; Selenoethionine; Flow injection; Atomic absorption spectrometry; Hydride generation; Microwave-induced plasma

1. Introduction

Selenium compounds have an ambivalent behav-ior in the environment. The availability of analyti-

∗ Corresponding author. Tel.: +81-298-52-1609;fax: +81-298-50-2574.E-mail addresses: amit.chatterje@nies.go.jp,chatterjeea@hotmail.com (A. Chatterjee).

cal techniques for the separation and determinationof selenium compounds at trace level has gainedconsiderable importance. Several selenoamino acidshave been reported to occur in organisms [1,2]. Aliterature search revealed that limited analytical workon selenoamino acid speciation has been carried outespecially with selenoethionine (Seet). Selenoethio-nine (ethyl(3-amino-3-carboxy-1-propyl)selenide), a

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0 0 0 3 -2 6 70 (01 )00970 -9

254 A. Chatterjee et al. / Analytica Chimica Acta 436 (2001) 253–263

dialkyl selenide (C2H5–Se–CH2–CH2–CH(NH2)-COOH), will carry a positive charge at relativelylow pH (localized to be protonated amino group),but will be zwitterionic (amino group, carboxylategroup) at intermediate pH, and become anionic (car-boxylate group) at higher pH [2]. The Seet has beentested for the ability to induce endogenous retro-virus expression in cultured AKR mouse embryofibroblasts [3] and found to be less toxic to the cellscompared to the selenomethionine (Semet) and otheramino acids [3]. The production of 1-methyladenine(1-MeAde) by gonad-stimulated substance (GSS)has also inhibited by Seet, a competitive inhibitorsof methionine [4]. This amino acid of Se is of sig-nificance only at high concentrations of selenium[1,5]. The increased use of Se as dietary supplemen-tation in animals and humans, methods are neededfor the determination of selenium compounds inurine, water and other environmental and biologicalsamples, so that, the metabolic fate and biogeochem-ical cycle of selenium is better understood. Methodsfor the determination of selenium compounds arereviewed by Pyrzynska [6] and others [1]. Popu-lar and reliable methods for the determination ofSeet and other Se compounds are AAS, AES, AFS,MIP-MS, ICP-MS and GC-ICP-MS [1,6–21]. Thedirect coupling of high-performance liquid chro-matography (HPLC) using several separation modeswith FAAS, HG-AAS, GFAAS, ICP-MS, MIP-MSand GC with ICP-MS detectors are the commonlyused methods for the determination Se compounds[7–22].

The hydride generation (HG) technique has becomea one of the useful alternative for the determination ofselenium as it increases transport efficiency (∼100%)and ultimately increases overall sensitivity. HG withflow injection (FI) is a more powerful tool for Sedetermination. The method is based on a reductionof the element from a higher oxidation state to thegaseous hydride with NaBH4. The sensitive tech-niques combined with a HG system are HG-AAS [1],HG-atomic fluorescence spectrometry (AFS) [15,22],HG-AES [15] and HG-ICP-MS [23]. However, onepotential drawback of HG is that it requires elementsto be in a particular oxidation state before a hydridemay be formed effectively [23]. This includes amongthe compounds in which the metalloid occurs in itslowest oxidation state, such as selenonium species

[1,24]. It is reported that unlike selenite, Seet doesnot directly form volatile selenium compounds in aHG system with NaBH4 and HCl [1,24]. Thus, fordetermination of Seet, a treatment prior to HG ismandatory. Papers containing different procedures fordecomposition of Seet to selenate and further reduc-tion to selenite are available [1,6]. The oxidation ofSeet to selenate using a microwave unit with K2S2O8[11], HBr–KBrO3 [25] and KBr–HCl [25] with UVirradiation are widely used for the decomposition, re-duction and estimation of Seet by FI-HG-AAS. Seet isalso determined by thermochemical HG-AAS [2,26].However, we have previously reported [1] that a num-ber of organicselenium compounds (seleno aminoacids, selenocholine, trimethylselenonium (TmSe)and dimethyl(3-amino-3-carboxy-1-propyl)seleniumiodide (DmpSe)) are HG active and generatedvolatile selenium compounds. We have also re-ported the direct estimation of TmSe in humanurine by FI-HG-AAS without any chemical pre-treatment before HG [27]. More recently we havecoupled the HPLC with the high power N2-MIP-MSfor the selenium speciation analysis [16]. The highpower N2-MIP-MS is more robust and used suc-cessfully for the determination of selenium com-pounds (selenite and selenomethionine) in urine[16].

For the current investigation, a direct FI-HG-AASmethod has been developed for the determinationof Seet with using NaBH4 and HCl. The developedmethod has excluded any chemical pretreatment priorto HG. Attention has been paid to measure the detec-tion limit, sensitivity, repeatability and reproducibilityof standard Seet, which is prepared in Milli-Q water.Finally, this HG system has been successfully coupledwith HPLC-N2-MIP-MS for the on-line separationand estimation of selenite and Seet (spiked) in thehuman urine.

2. Experimental

2.1. Instrumentation

2.1.1. Atomic absorption spectrometerA Perkin-Elmer Zeeman 5100 (with D2 background

corrector) flame atomic absorption spectrometer fit-ted with electrically heated quartz T-tube served as

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Fig. 1. Schematic diagram of HPLC-HG-N2-MIP-MS/QTAAS.

the selenium specific detector. The selenium com-pounds were reacted with HCl in the reaction coil(Fig. 1) of Perkin-Elmer FIAS 100 system equippedwith a gas–liquid separator and a Perkin-Elmer AS91 autosampler. The mixture solution (HCl with Secompounds) was moved to gas–liquid separator witha 300 mm long PTFE (0.75 mm id) tubing and thenreacted with the alkaline sodium tetrahydroborate(Fig. 1). The gaseous products thus formed wereseparated from the liquid in the gas–liquid separatorand flushed into the quartz tube by a stream of nitro-gen (Fig. 1) with a 400 mm long PTFE (1.0 mm i.d.)tubing. The Perkin-Elmer selenium electrodeless dis-charge lamp (EDL) was (part no. N305-0672, serialno. 1209 M676) operated at 210 mA. The operationconditions for FI-HG-AAS are summarized in Table 1.

Table 1Operating conditions for FI-HG-AAS

Atomic absorption spectrometer Perkin-Elmer 5100Lamp current (EDL) 210 mAWavelength 196.0 nmElectrically heated quartz T-tube temperature ∼830◦CNaBH4 (0.3% in 0.2% NaOH) 4.5 ml min−1

Carrier flow rate (3.0 M HCl) 5.6 ml min−1

Sample loop 500 �lFill time 10 sInject time 15 sNitrogen gas flow 114 ml min−1

A schematic diagram of the complete instrumentalsetup used and the on-line HPLC connection detailsare shown in Fig. 1. Conventional background cor-rector of the AAS was used. Inherent backgroundcorrected Se signals and background signals wereread into a personal computer via the Perkin-ElmerGEM software. More details about AAS and thebackground correction were described previously[1,2,27]. Furthermore, blank was measured and blanksubtraction was carried out during data processing.

2.1.2. ChromatographyThe high-performance liquid chromatography sys-

tem (HPLC) consisted of a Perkin-Elmer Model Series410 B10 solvent delivery unit (Perkin-Elmer, Nor-walk, CT, USA) and a Rheodyne 9725 six-port injec-tion valve (Cotati, CA, USA) with a 100 �l injectionloop. A GS-220 (gel-permeating, 7.6 mm × 500 mm:Asahi Kasei, Japan) was used for separation study.The column was equilibrated by passing at least100 ml (flow rate 1 ml min−1) of the mobile phasethrough the column before any injection of the se-lenium compounds. The outlet of the column wasconnected to one-side of a T-connector (Omnifitthree-way 0.8 mm bore size T-connector, cat. no.002401) via a 300 mm × 0.25 mm i.d. polyether etherketone (PEEK) capillary tubing (Fig. 1). The HCl car-rying tube from the FIAS-100 system was connectedto the second end of the same T-connector (Fig. 1).

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The mixture solution departing from the last end ofthe T-connector was connected with the gas–liquidseparator of the FIAS-100 HG system (Fig. 1; sameHG system with identical analytical conditions usedfor AAS, Perkin-Elmer HG system; Table 1) with a300 mm long PTFE (0.75 mm i.d.) tubing. The out-let of the HG system was connected with 400 mmlong PTFE tubing (1.0 mm i.d.) to the sample inletof the nebulizer of MIP-MS (Fig. 1). The ion in-tensities at m/z = 80 (80Se) was recorded with thetime-resolved analysis software© Version (Hitachi,Japan). For quantification, the chromatograms wereexported, peak areas were calculated, and the con-centrations were determined with external calibrationcurves established for each of the Se compounds andwith standard addition technique.

2.1.3. Nitrogen microwave-induced plasma massspectrometry (N2-MIP-MS)

A Hitachi P-6000 N2-MIP-MS (Hitachi, Ibaraki,Japan) was used for this study. In Table 2 the oper-ating conditions of N2-MIP-MS are displayed. Themicrowave power (2.45 GHz, 1.5 kW max) was pro-duced by a magnetron (H3862: Hitachi Ltd.) operatedby a dc power supply and fed to a cavity known

Table 2Operating conditions for N2-MIP-MS

Frequency 2.45 GHzMicrowave power

Forwarded 1.3 kWReflected <40 W

Plasma gas flow 15 l min−1

Carrier gas flow 1.1 l min−1

Peak point/mass 10Dwell/time 2.0 msNumber of sweeps 1500 timesNebulizer (Meinhard) ConcentricTemperature of spray chamber <5◦CSampling cone (Pt) 0.8 mm orificeSkimmer cone (Pt) 0.4 mm orificeSample uptake rate 1.5 l min−1

Measuring parametersMonitored signals 80Se+ m/z = −80

Total analysis time 1600 sMass analyzer Quadrupole analyzer: QMG420-4 Balzers, Liechtenstein; Mo rods,

200 mm length, 8 mm diameter, radio frequency of 2.25 MHzIon detector Channeltron electron multiplier 4831 G mounted on a quadrupole

analyzer with 90◦ ion deflection and off axisPulse counting Pulse amplifier C3866 Hamamatsu Photonics, resolution 10 ns, maximum counting rate 107 cps

as Okamoto cavity through a rectangular waveguide(WRJ-2). The cavity was cooled by circulating coldwater (20◦C) from a refrigerator. An annular-shapedelectric field, where the microwave discharge main-tained, was produced between an inner conductor andan outer cylindrical conductor terminated by a frontplate. A quartz discharge tube of 10 mm i.d. (1 mmthickness) was inserted into an inner conductor. Theplasma was ignited by a Tesla coil. A stable, an-nular (doughnut) shape, pink color nitrogen plasmawas produced. Details about the quartz dischargetube were described earlier [16,28]. A Meinhardconcentric-type nebulizer (Hitachi Electric Co. Japan,part nos. P97M170, 300-8350) connected with PTFEsampling tubing (Hitachi, part no. 300-8868) withouta desolvation system was used for the sample intro-duction system. The outlet of the HG system wasdirectly connected to the sample inlet position of thenebulizer. A Neslab refrigerating circulator was usedto maintain the temperature of the glass spray cham-ber at 5◦C. More detail descriptions of the microwavesystem, plasma ignition system, sample introductionsystem, MS instrument and the interface system waspublished previously [16]. The schematic diagram ofthe HPLC-HG-N2-MIP-MS is shown in Fig. 1.

A. Chatterjee et al. / Analytica Chimica Acta 436 (2001) 253–263 257

2.2. Chemicals and reagents

Urine was collected from one of the volunteersof our group. All reagents were of analytical gradeand used without further purification. Sodium sele-nate (No. 71948) was purchased from Fluka, sodiumselenite pentahydrate from Merck (No. 106607) andseleno-dl-ethionine from Sigma (S 3750). Stock so-lutions were prepared in Milli-Q water (18.2 M� cm,Milli-Q SP, TOC; reagent water system, Nihon Milli-pore Ltd. Yonezawa, Japan) from anhydrous sodiumselenate (1196 mg to 500 ml, 1000 �g Se ml−1), fromsodium selenite pentahydrate (1666 mg to 500 ml;1000 �g Se ml−1), and from Seet (53.2 mg to 20 ml,1000 �g Se ml−1). The stock solutions were storedin refrigerator at −20◦C before use. No degradationof the compounds was observed over 3 months ofstorage [1,2]. Solutions of the selenium compoundswith concentration in the range 1.00–20.0 ng Se ml−1

were prepared by appropriate dilution of the stocksolutions with Milli-Q water. Sodium tetrahydrob-orate solutions (0.2–1.5%, m/v) were prepared bydissolving NaBH4 powder (Merck, No. 1063710) of96% purity in Milli-Q water and stabilizing the solu-tions with sodium hydroxide (0.2%, m/v; Merck, No.71690). The solutions were filtered through beforeuse to eliminate turbidity and then stored in a refrig-erator. Hydrochloric acid solutions (2.0–6.0 M) wereprepared by dilution of concentrated HCl (Merck,No. 100319, further purified in a quartz sub-boilingdistillation unit). Mobile phase for the gel-permeatingchromatography was prepared by dissolving 22.79 gof 10% tetramethylammonium hydroxide (25 mM;Nacalai Tesque, Inc., Kyoto, Japan) with 2.6015 gmalonic acid (25 mM; Nacalai Tesque, Inc., Kyoto,Japan) in water and adjusting the pH to 7.5 by addi-tion of 2.0 M aqueous NH3 solution (Merck).

3. Results and discussion

3.1. Optimization of experimental parameters usingFI-HG-AAS

The following chemical and physical parameters areoptimized to achieve the best analytical performanceof the FI-HG-AAS system, for the quantification ofSeet. Peaks are always recorded as peak area because

Fig. 2. Dependence of signal intensity of Seet on the concentrationof NaBH4 (mean ± S.D.; n = 5; 20.0 ng Se ml−1; 3.0 M HCl;N2 flow 114 ml min−1; injection volume 500 �l; quartz furnacetemperature 830◦C).

that has improved the signal-to-noise ratio by a factorof ∼5 compared to peak height.

3.1.1. Sodium tetrahydroborate concentrationThe dependence of the signal intensities of Seet

(15.9 ng Se ml−1) on the NaBH4 concentrations(0.3–1.5% NaBH4 in 0.2% NaOH) in the flow modehas been investigated. The results indicate that thesignal intensities increase with increasing NaBH4concentration (Fig. 2) and is maximum at 0.3%NaBH4. Above 0.3% NaBH4 the signal intensitiesare decreased, baseline is unstable and backgroundsignal’s repeatability is low (R.S.D. = 13%, n = 5)in FIAS-100 system. Some times an irregular doubletpeak has been formed. The inability of the gas–liquidseparator to deal with increased concentrations ofNaBH4 resulted in a worse performance and loss ofsensitivity. The liquid has been overflowed from thegas–liquid separator above 0.7% NaBH4. Workingwith such high concentration (1.0–1.5%) of NaBH4 isalso impracticable due to excessive background noise,which has been generated from the large amount ofhydrogen that is produced by reaction of NaBH4 withHCl. So 0.3% NaBH4 has been used as the optimumcondition for the FI-HG system.

3.1.2. AcidityThe signals for hydride forming elements are

known to be dependent on the concentration of H+ions. So the efficiency on the generation of volatileselenium compound in various HCl (2.0–6.0 M) solu-tions has been investigated. The most sensitive condi-tion is 3.0 M HCl (Fig. 3). Small changes around theoptimum acidity cause an apparent decrease in the

258 A. Chatterjee et al. / Analytica Chimica Acta 436 (2001) 253–263

Fig. 3. Dependence of signal intensity of Seet on the concentrationof HCl (mean ± S.D.; n = 5; 20.0 ng Se ml−1; 0.3% NaBH4 in0.2% NaOH; N2 flow 114 ml min−1; injection volume 500 �l;quartz furnace temperature 830◦C).

signal intensities. With further increase in HCl con-centrations, peak area and height decrease sharplywith decreasing baseline noise.

3.1.3. NaBH4/HCl flow ratioThe NaBH4/HCl solution flow ratio influenced the

volatile selenium compounds generation. An optimumsignal has been obtained when the flow ratio is 0.80. Afurther increase in the flow ratio decreases the signalintensity, enhances the baseline noise and the liquidoverflows from the gas–liquid separator, that has beenobserved in our previous investigations [1,27] also.

3.1.4. Carrier gas flowThe flow rate of argon that is needed as carrier gas

to transport the hydrides form in the gas–liquid separa-tor to the quartz-tube (QT) is an important parameter.With increasing Ar flow from 72.5 to 316 ml min−1

the analyte signal increases up to 114 ml min−1 andthen decreases with increasing Ar flow rate (Fig. 4).

Fig. 4. Dependence of signal intensity of Seet on the flow rateof argon (mean ± S.D.; n = 5; 20.0 ng Se ml−1; 0.3% NaBH4 in0.2% NaOH; 3.0 M HCl; injection volume 500 �l; quartz furnacetemperature 830◦C).

Fig. 5. Dependence of signal intensity of Seet on the temperatureof the quartz tube atomizer (mean ± S.D.; n = 5; 20.0 ng Se ml−1;0.3% NaBH4 in 0.2% NaOH; 3.0 M HCl; injection volume 500 �l;N2 flow 114 ml min−1).

An optimum 114 ± 5 ml min−1 has been used for themeasurements. With increasing gas flow rate further(up to 316 ml min−1) the Seet signal repeatability isimproved, the background noise also decreased. But,frequently irregular doublet peak is appeared.

3.1.5. Quartz tube temperatureOn increasing the quartz furnace temperature

(700–950◦C) the signal intensity increases to a maxi-mum at 830◦C (Fig. 5). A further increase in tempera-ture causes a decrease in signal, with increasing base-line noise. So, 830◦C has been used as the optimumtemperature (maximum signal) for the measurement.However, a temperature of 900◦C has been recom-mended by the manufacture of the atomic absorptionspectrometer for selenium measurement. A loss of∼30% of the signal intensity at 900◦C compared withthe optimized temperature of 830◦C at the quartz tubeatomizer is observed (Fig. 5). Moreover, the peak areaand height have a good repeatability (R.S.D. = 4.3%,n = 5) at temperature higher than 900◦C.

3.2. Performance

The performance of the FI-HG-AAS system is char-acterized by the linearity of the standard curves andlimits of detection (based on 3σ for the blanks). Theabsolute detection limit of Seet based on the variabil-ity of the reagent blank (3σ , n = 20) is 0.12 ng; thesensitivity is 0.05 (peak area per ng of Se ml−1). Thequantification limit based on the concentration (ng ofSe ml−1) corresponding to 10σ of the blank (n = 20)is 0.85 ng of Se ml−1 Calibration graph is obtainedfrom the areas of the HG-AAS signals (seven repli-cates) with standard solutions of Seet at concentrations

A. Chatterjee et al. / Analytica Chimica Acta 436 (2001) 253–263 259

0.00, 1.00, 5.00, 10.0, 15.0 and 20.0 ng Se ml−1 Thegraph is linear in this concentration range. The rela-tive standard deviation (R.S.D., n = 7, repeatability)of the peak areas do not exceed 9.5% even at the low-est concentration of 1.00 ng Se ml−1 of Seet. At con-centration of 20.0 ng Se ml−1 highest relative standarddeviation is 2.8%. The reproducibility, determined forthe peak areas (5.05 ng of Se ml−1) by calculating therelative standard deviation of three analyses performedon the three different days is 9.2%. The regressioncalibration equation is y = 0.0384x + 0.0093, with acorrelation coefficient of 0.9998, where x representsanalyte mass (ng) and y is the integrated absorbance(s), and y = 0.0056x + 0.0052 with a correlation co-efficient of 0.9999, where x is the analyte mass (ng)and y is the peak height (absorbance). So peak heightmeasurement is also applicable for analysis.

3.3. Interference and recovery study

The interference of selenite and selenate (1.00–20.0ng of Se ml−1 each) with respect to Seet has been stud-ied under the experimental conditions employed forSeet (0.3% NaBH4 in 0.2% NaOH and 3.0 M HCl). Nosignal has been found from selenate in the HG system.But, selenite gives a strong signal with a linear calibra-tion graph for 1.00–20.0 ng Se ml−1. The recovery ofSeet (20.0 ng Se ml−1) has been studied in the presenceof selenite and selenate (20.0 ng Se ml−1 each) underthe same analytical conditions employed for Seet. Re-covery of Seet is 98–105% in the presence of selenate.So selenate does not interfere with Seet and is unableto form volatile compounds with 0.3% NaBH4 and3.0 M HCl, as we observe previously [1,6,27]. But se-lenite has a strong positive interference. Both, seleniteand Seet are generated hydride active Se-compoundsunder the present analytical conditions. Moreover, thehydride active compounds formation and atomizationefficiencies of selenite is found to be about 4.0 ± 0.5times higher than Seet under the present experimentalconditions.

3.4. Chromatographic separation and determinationof selenite and Seet in human urine byHPLC-HG-N2-MIP-MS

However, it is found that selenite present either inwater or in urine has strongly interfere with Seet [1].

Further, even a small amount of selenite present in theenvironmental samples is a serious potential source oferror for Seet measurement under the current analyti-cal conditions. Therefore, selenite should be separatedfrom Seet before quantification. Due to lack of timeresolve analysis software in our AAS instrument, weare unable to couple HPLC chromatographic columnon-line with the HG-AAS. However, we have success-fully coupled the same HG system (having identicalHG conditions used as those for the AAS in termsof gas and reagents flow rates and concentration ofreagents) with the HPLC-N2-MIP-MS for the sepa-ration and determination of selenite and Seet in thehuman urine. It has been well known that chromato-graphic columns decrease the interference problemsin the urine [2,6]. So, we have used the HPLC sys-tem to minimize the matrix interference that is presentin the human urine. The MIP-MS is used because (i)as far as our knowledge, the separation and determi-nation of Seet by HPLC-HG-N2-MIP-MS is not wellreported, (ii) the major isotope of Se (m/z = 80) isused for the ions signals measurement, as there is nospectroscopic interference related to 40Ar2

+, whichis very prominent in the ICP-MS [16,28]. The spec-troscopic interference in the m/z region for other se-lenium isotopes is also not found since nitrogen isused as the plasma gas [16,28] in the MIP. Efficientchromatographic systems are reported for the sepa-ration of selenous acid and Seet with using differ-ent kinds of the mobile phases [7,28,29], which aresuitable for the ICP-MS. We have used cationic, an-ionic, reversed-phase and gel-permeating chromato-graphic conditions to observe the HG efficiency ofSeet with different mobile phases (Table 3). Differ-ent mobile phases have suppressed the volatile hy-dride formation from Seet (Table 3). Moreover, withthe 15–30 mM phosphate buffer, the suppressing ofthe volatile hydride formation from Seet is minimal.The recovery of Seet (101–105%) that is prepared inthe phosphate mobile phase is almost same as that ofthe Seet, prepared in Milli-Q water (Table 3). The se-lenium compounds are present in the solution as an-ions, cations or zwitterions. The retention behavior ofselenous acid and Seet on the anion-exchange columnis governed by the pH-controlled protonation of sele-nium oxo-anions and phosphate anions. These anionscompete for the ammonium groups of the stationaryphase in the PRP-X100 anion-exchange column. We

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Table 3Percentage recovery of selenoethionine (Seet) in different mobilephases

LC 1a LC 2b LC 3c LC 4d LC 5e

Seet (%) 100 101–105 70–74 2.3–4.0 14–27

a Milli-Q water.b 15 mM phosphate buffer at pH 7.0 for the PRP-X100

anion-exchange column [16,29].c 25 mM tetramethylammonium hydroxide + 25 mM malonic

acid at pH 5.0 for the Asahipak GS220 gel permeating column [7].d 20 mM pyridine buffer at pH 2.6 for the Supilcosil LC-SCX

cation-exchange column [28].e 10 mM tetraethylammonium hydroxide+4.5 mM malonic acid

at pH 6.8 in 0.05% methanol for the Inertsil ODS reversed-phasecolumn [28,30].

have tested the phosphate mobile phase with varyingconcentrations (15–30 mM) at different pH conditions(5.4, 6.0, 6.7 and 7.2) in the PRP-X100 column. How-ever, the Seet is not separated well with phosphate mo-bile phase in the PRP-X100 anion-exchange column.A broad peak of Seet is formed after 600 s with usingthe phosphate mobile phase. The base line has beenincreased after 600 s. Retention of Seet in PRP-X100column may be ascribed due to the high hydropho-bic interaction of Seet with the stationary phase of thecolumn. Further, phosphate mobile phase, which hasless hydrophobic character than the stationary phase(styrene–divinylbenzene copolymer) of the column,reduces slowly the hydrophobic interaction betweenthe column material and Seet. So Seet is eluting slug-gishly after 600 s. The GS-220 gel-permeating col-umn with 25 mM tetramethylammonium hydroxide +25 mM malonic acid buffer at pH 7.5 is suitable forthe separation of Seet from the selenite and Semet(Fig. 6). Under these conditions we are able to obtaina good resolution of the peaks in a reasonable time(Fig. 6). This can be attributed to the different interac-tion processes between the stationary phase and sele-nium compounds at the pH chosen. Although, GS-220is made of polyvinyl alcohol, a neutral and hydrophiliccompound, it has both negative charge and hydropho-bicity character. Negative charge comes from carboxylgroups, which have been introduced during polymer-ization procedure on the resin while hydrophobicitycomes from double bonds, added to harden the resin inorder to resist mechanical stress during HPLC stage.These two help to improve separation of various com-

Fig. 6. Chromatogram obtained with a solution of selenite(25.5 ng ml−1 Se), selenomethionine (Semet; 49.2 ng ml−1 Se) andselenoethionine (Seet; 100.6 ng ml−1 Se) in distilled water on aGS-220 gel-permeating column with HG-N2-MIP-MS (mobilephase 25 mM tetramethylammonium hydroxide + 25 mM mal-onic acid buffer at pH 7.5, injection volume 100 �l, flow rate1.0 ml min−1).

pounds. In other words, GS-220 separate compoundsbased on not only the size of the molecule but also thecharge-state and hydrophobicity. Selenate at pH 7.5carries two negative charges. So, it is repelled by thenegatively carboxylic group of the resin and elutes inthe front of the chromatogram. Increasing pH, repul-sion between carboxylic group and selenate may in-crease [30]. Hence, this anionic repulsion may be re-sponsible for decreasing the retention time of selenatewith increasing pH values [30]. Selenous acid is about22% mono-negatively and about 78% di-negativelycharged at pH 7.5. Both anionic repulsion and hy-drophobic interaction with stationary phase may be ac-countable for the weak retention of this compound onthe column, and elutes after the selenate. Semet at pH7.5 exists almost as a zwitterionic (ammonium groupand carboxylic group, about 4% L−, 96% HL), whichdoes not interact with negatively charged carboxylicgroup of the resin but may have a strong hydrophobicinteraction. This hydrophobic interaction favors Semetto retain in the column, and elutes after selenite and se-lenate but have shorter retention time than Seet, whichis more hydrophobic compared to Semet. Hence, theSeet is eluted from the GS-220 gel-permeating columnafter Semet (Fig. 6). Fig. 7 shows the selenium speciespresent in the urine under above analytical separationconditions. Peaks have been identified according to

A. Chatterjee et al. / Analytica Chimica Acta 436 (2001) 253–263 261

Fig. 7. Chromatogram obtained for (a) the human urine and (b)human urine spiked with Seet (97.7 ng ml−1 of Se) on a GS-220gel-permeating column with HG-N2-MIP-MS (conditions as inFig. 6).

the retention times (Fig. 7) with those of the authen-tic standards (Fig. 6) and confirmed by spiking withthe standard selenium compounds those are preparedin Milli-Q water. The selenite and two unknown sele-nium compounds peaks are detected (Fig. 7). The se-lenite, estimated using standard addition technique, is3.6±0.2 �g l−1 in agreement with the HPLC-ICP-MS(3.8 ± 0.23 �g l−1). The peak of Seet is not found inthe urine. Spikes of different Seet concentrations areadded to the urine (25.0, 49.4, 98.0 and 198 ng ml−1).The Seet is successfully separated from the urine ma-trix (Fig. 7). The percentage recoveries of Seet arein the range of 93–102%. As a result, with using thechromatographic system the matrix interference areeliminated in the urine. The selenate (most probable inthe urine) is not interfering, as selenate is HG inactivewith the present HG conditions. Thus, the developedchromatographic system is adequate for the separa-tion of Semet, Seet and selenite and we have appliedsuccessfully for the estimation of above compoundsin the human urine.

Several authors have noted the interference prob-lems; mainly VIIIB and IB elements produce theinterference problems in the HG system for determina-tion of Se [31,32]. It has been noted that the seleniumcannot be detected in the HG system in the presenceof high concentration of Cu and Ni [31]. At 1000-foldexcess of Co, the response of Se is dropped to 20%[31]. The mechanism of these interferences is still the

subject of considerable discussion [31]. We have ob-served the signal suppressions for the direct (withoutHPLC) determination of selenous acid and Seet, byour optimized HG-AAS system, from tap water andurine. Surprisingly, the interference effect is negligi-ble when we have used the HPLC system. This is dueto the utilization of the GS-220 chromatographic col-umn. This column is capable to separate the interfer-ing metals from Se compounds. Interfering elementsare eluting out in the solvent fronts with the mobilephase and minimized the HG suppression problems.

Sodium tetrahydroborate is a versatile reagent,which is widely used for its reducing and hydridetransfer properties [33]. It is used for conversion ofelements species present in aqueous solution intovolatile hydrides. In this role, tetrahydroborate reagentcan act as a reductant and as a hydride source. In itsreaction with selenium oxy-anion, which contains se-lenium in tetravalent oxidation state, the reaction withtetrahydroborate takes selenium compound throughthe corresponding selenide:

OSe(OH)2 + BH4− + H+ → SeH2 + H3BO3 + H2

The reaction between NaBH4 and an ion in solutionis sensitive to pH, and it appears that, for the rapid re-action, target species must not be present in solutionas a negatively charged species. As pK1 for selenousacid is around 2.5, which is fully protonated belowpH 2. Hence, reaction must be carried out at very lowpH and we have used 3 M HCl [33]. Under this condi-tion, selenous acid reacts with NaBH4 and reduces toselenide. Further, selenic acid is not fully protonatedat 3 M HCl (pK2 = 2.0 implying a pK1 value of ap-proximately −3). Hence, selenate is inactive in HGsystem and tallies well with our experimental finding.HG forming capabilities of Seet may be described insimilar way as selenite, because Seet may have simi-lar pK values (not reported), and behaving in similarway as Semet (pK1 = 2.28 and pK2 = 9.21). So,in 3 M HCl, it may be protonated. Under these con-ditions Seet is reacted with NaBH4, decomposed andproduced ethylhydrogenselenide, which dimerized tovolatile diethyldiselenide. This diethyldiselenide hasbeen transported to the quartz atomizer by carrier gas.There, it has been decomposed to Se atoms and gener-ated signal intensity. Hence, Seet is HG system active.

The direct coupling of HPLC with ICP-MS detec-tion seems to be one of the well known and mostly

262 A. Chatterjee et al. / Analytica Chimica Acta 436 (2001) 253–263

used methods for the speciation of Se. It is easily cou-pled with HPLC. But the performance of ICP-MS isrestricted, the sensitivity for selenium compounds iscomparatively low due to its high first ionization po-tential, and for all Se isotopes the determination suf-fers from interferences, isobaric as well as polyatomic[17]. The polyatomic interferences such as 40Ar2

+ atm/z = 80, can be overcome using cold plasma tech-nique [34] and application of collision and reactioncells [35]. However, the cold plasma technique is notsuitable for Se analysis and for high matrix containingsamples, because of low plasma, electron and ioniza-tion temperatures that causes low ionization of Se inthe plasma [17,36]. The GC-ICP-MS coupling usuallyprovided better resolved peaks and lower detectionlimits than HPLC-ICP-MS, but the absence of reliableand commercially available transfer lines to coupledGC efficiently with quadrupole-based ICP-MS toavoid condensation is till in the developing conditions[17]. Due to non-volatile nature of Seet, derivatizationwith suitable organic compounds are essential priorto GC injection. The sample preparation steps are te-dious and time consuming [17]. During derivatization,species transformation and by-product can be formeddisturbing the rapid identification and correct quan-tification of the sought main compound. As far as ourknowledge, the coupling of GC with a sector-basedICP-MS is not well reported. So, the Ar-related poly-atomic interference is common and severe in theGC-ICP-MS also. High resolution ICP-MS is themost effective solution for removing the argon relatedpolyatomic interferences. But with increasing reso-

Table 4Detection limits (DLs) of Seet in different spectroscopic techniques

Methods DLs (�g l−1) Reference

HPLC-microwave digestion -HG-QTAFS 0.9 [20]Capillary electrophoresis-UV detector 164 [39]Quartz thermochemical HG-AAS 1.4a [26]HPLC-offline-ETAAS 5.0 [11]HPLC-FAAS 100 [40]HPLC-ICP-MS 1.0 [40]HPLC-GFAAS 10 [40]HPLC-ICP-AES 100 [11]HPLC-high resolution-ICP-MS 0.87a [19]HPLC-hexapole collision and reaction cell ICP-MS 0.21 [38]HPLC-HG-MIP-MS 0.97 This workHG-QTAAS 0.12a This work

a Absolute detection limit (ng).

lution (resolution for 40Ar2+ and 80Se+ is 9688) the

detection capability decreases. For the high-resolutionmode the absolute transmission of the system may belowered by a factor of 10–100. So the detection capa-bility in the high-resolution mode is about 2 orders ofmagnitude lower [37]. In some applications, such asthe speciation of selenium in biological materials, thehigh sensitivity required could not be achieved withhigh resolution ICP-MS [38]. However, the cost ofthe high resolution ICP-MS is about two to four timeshigher than the normal quadrupole-based ICP-MS.But, with HPLC-HG-N2-MIP-MS, we efficientlymonitor the 80Se+ (m/z = 80) ion signals. So, the Arrelated polyatomic interference is not observed at all[16,28]. Matrix related interferences such as ArCl+and BrH+ (m/z = 77, 80 and 82), very common inurine matrix for Ar-ICP-MS, are not found also as wehave used the HG system [16]. Moreover, the gas run-ning cost of the high power N2-MIP-MS is lower thanthat of the Ar-ICP-MS. Additionally, a high-puritynitrogen gas supply minimizes contamination of theplasma, and is easily obtained from liquid nitrogenboil-off. So, the background noise is low. Hence, thedetection limits obtained with the current method arecomparable with other spectroscopic methods (Ta-ble 4). Using HPLC-HG system most of the matrixinterferences are removed. So, the detection limit forSeet is remained almost same in urine as that of Seetprepared in Milli-Q water. Hence, the method is verypromising for the determination of Seet in real sample.

In summary, as far as our knowledge, this is thefirst report of the direct determination of Seet in

A. Chatterjee et al. / Analytica Chimica Acta 436 (2001) 253–263 263

urine by using HPLC-HG-N2-MIP-MS. The decom-position of Seet to selenate/selenite is mandatorybefore HG and has complicated its measurement.The current developed method directly determinedSeet from samples by HG-MIP-MS/AAS that elim-inates the error caused by pretreatment step and/orindirect methods. It is reported that Seet is almostresistant to acid attack [2]. Complete decompositionis possible using nitric–sulfuric–perchloric acid di-gestion at 310◦C. Br2/HBr could decompose manyorganic selenium compounds, including Seet to se-lenite, the form necessary for a HG system [2,6]. Thepresent developed method is safe as it excludes allpretreatment (UV/microwave/acids) prior to HG. Sothe developed method decreases sample and reagentconsumption, waste generation and risk of contam-ination, with a reasonable detection limit. Applyingthe HPLC-HG-N2-MIP-MS and both selenite andSeet (spiked) in human urine have been separated anddetermined adequately.

Acknowledgements

The authors gratefully acknowledge JISTEC, JST,STA, Japan, CSIR India, for financial support; Dr.J. Yoshinaga for instrumental facility; Mr. MinoruYoneda for data transformation and Dr. Sukti Hazrafor helpful comments during the preparation of themanuscript. Amit Chatterjee heartily acknowledgesMrs. Shizuko Kinoshita for her technical support.

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